CN106674368B - A kind of preparation method of Phellinus fructification cell wall activity Thick many candies - Google Patents
A kind of preparation method of Phellinus fructification cell wall activity Thick many candies Download PDFInfo
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- CN106674368B CN106674368B CN201611187085.4A CN201611187085A CN106674368B CN 106674368 B CN106674368 B CN 106674368B CN 201611187085 A CN201611187085 A CN 201611187085A CN 106674368 B CN106674368 B CN 106674368B
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- 238000004458 analytical method Methods 0.000 description 3
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- 239000002244 precipitate Substances 0.000 description 3
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- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- 241001556385 Sanghuangporus baumii Species 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
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- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
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- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 101000729818 Bacillus licheniformis Glutamate racemase Proteins 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- LKDRXBCSQODPBY-VRPWFDPXSA-N D-fructopyranose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-VRPWFDPXSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
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- 239000002893 slag Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Sustainable Development (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a kind of preparation methods of Phellinus fructification cell wall activity Thick many candies; include the following steps: the processing Step 1: fructification residue; Step 2: the ultra-fine grinding of Phellinus residue and Step 3: Phellinus fructification cell wall activity Thick many candies extraction; the raw material wherein used in the step 1 mentions the residue of intracellular polyse for fructification water, and the step 3 uses aqueous extraction-alcohol precipitation technology；The preparation method further includes the steps that Phellinus fructification cell wall activity Thick many candies are further purified；The invention further relates to a kind of applications using the resulting Phellinus fructification cell wall activity Thick many candies of above-mentioned preparation method.Compared with prior art, the residue after preparation method of the present invention uses Phellinus fructification to extract realizes waste cycling and reutilization for raw material；And active Thick many candies prepared by the present invention are stable macromolecular polysaccharide, and its content is higher, has good immunocompetence.
Description
Technical field
The present invention relates to Edible mushroom processing fields, and in particular to a kind of preparation of Phellinus fructification cell wall activity Thick many candies
Method.
Background technique
Phellinusbaumii (Phellinus baumii Pilat), is commonly called as Phellinus, is a kind of perennial, filemot treasure
Expensive medicinal fungi has the laudatory title of " forest gold ".Phellinus have it is antitumor, improve immune, anti-aging, protect liver, analgesia etc.
Effect, is used widely in functional food and clinically.
As rare medicinal fungi, the main active of Phellinus is polysaccharide, at present the research to Phellinus polysaccharide both at home and abroad
With using water-soluble intracellular polyse and its anticancer effect is concentrated mainly on, relevant preparation process is existing much to be reported.But at present
The extraction yield of conventional water grape entity intracellular polyse is very low, and probably in 1%-2% or so, a large amount of polysaccharide also remains in son
In solid-cell wall.It is considerable that water mentions the Phellinus level of residue after intracellular polyse, cell wall polysaccharides rich in, this part is more
Sugar usually not continues extraction and application and is dropped, and has very big researching value, therefore it is living to extract Phellinus fructification cell wall
Property polysaccharide is good developing direction；On the other hand, the product of Phellinus polysaccharide is mainly that conventional water extract-alcohol precipitation obtains both at home and abroad at present
The fructification intracellular polyse arrived, and polyoses content is not high, only 30% or so, molecular weight is concentrated mainly on hundreds of thousands to 1,000,000
Between.
Chinese patent CN103319618A discloses a kind of preparation method of active polysaccharide of male agaric mycelium comprising step
Rapid: the polyoses content of the extraction of cultured mycelia, mycelium polysaccharides, preparation is lower, and specially 16~38%.
Therefore, sub- solid-cell wall middle-molecular-weihydroxyethyl is excavated in 2,000,000 or more macromolecular polysaccharide, facilitates treasure
The further development and utilization of dilute Resources of Medicinal Fungi.
Summary of the invention
The purpose of the present invention is to overcome the defects in the prior art, and it is thick more to provide a kind of Phellinus fructification cell wall activity
The preparation method of sugar, the preparation method is simple, prepare that resulting activity Thick many candies content is higher, and molecular weight is larger.
To achieve the above object, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of preparation method of Phellinus fructification cell wall activity Thick many candies, including walks as follows
It is rapid:
Step 1: the processing of fructification residue: the residue that fructification water mentions intracellular polyse being crushed with medicinal herb grinder, mistake
Sieve.Add boiling water after water soak at room temperature to extract repeatedly, discard Aqueous extracts, to remove water-soluble intracellular polyse.After inciting somebody to action repeatedly water extraction
Phellinus residue drying, it is spare.
Step 2: the ultra-fine grinding of Phellinus residue: using and pulverize vibrating grinder for the resulting Phellinus residue of step 1
It crushes, sieving.
Step 3: the extraction of Phellinus fructification cell wall activity Thick many candies: the Phellinus residue after step 2 is pulverized
Middle addition water, soak at room temperature add distilled water boiling waterbath, and filter residue repeats to extract, combined extract；Subtract on a rotary evaporator
Pressing concentrated extracting solution to material ratio is 1:1.2-1.5, prepares concentrate；Take concentrate centrifugation removal insoluble impurities, leave and take from
Heart liquid；In centrifugate plus ethanol precipitation, sediment separate out carry out sediment using alcohol identical with ethanol precipitation concentration
It washs repeatedly, then dissolves sediment using water, carry out the small-molecule substance in dialysis removal sediment with bag filter, it is finally right
The sediment of removal small-molecule substance is freeze-dried, to obtain Phellinus fructification cell wall activity Thick many candies.
In order to further optimize the above technical scheme, technical measures adopted by the present invention further include:
Preferably, the preparation method of Phellinus fructification cell wall activity Thick many candies of the present invention further includes to Phellinus
The step of solid-cell wall activity Thick many candies are further purified.
Preferably, the mesh number being sieved in the step 1 is 20 mesh, and the mesh number being sieved in the step 2 is 200 mesh.
Preferably, in the step 2 pulverize vibrating grinder operation temperature be -10 DEG C, the operating time be 10~
20min, preferably 15min.
Preferably, the concrete operations parameter of the step 3 is as follows: adding in the Phellinus residue after step 2 is pulverized
Entering the water of 10-15 times of weight, soak at room temperature 30-60min adds distilled water boiling waterbath 1-3h, and filter residue repeats to extract 2-3 times,
Combined extract；It is 1:1.2-1.5 that extracting solution to material ratio is concentrated under reduced pressure on a rotary evaporator, prepares concentrate；Take concentration
Liquid, 8000-12000 × g are centrifuged 15-30min and remove insoluble impurities, leave and take centrifugate；Add ethanol precipitation 20h in centrifugate
More than, sediment separate out washs sediment using alcohol identical with ethanol precipitation concentration repeatedly, then using water-soluble
Sediment is solved, with 8000-10000 molecular weight bag filter dialysis 2-4d, the small-molecule substance in sediment is removed, finally to removal
The sediment of small-molecule substance is freeze-dried, to obtain Phellinus fructification cell wall activity Thick many candies.
Preferably, for the concentration of alcohol that the ethanol precipitation uses for 20%-80%, more preferable concentration of alcohol is 20-60%,
Further preferred concentration of alcohol is 20-25%.
Preferably, the content of the Phellinus fructification cell wall activity Thick many candies of the freeze-drying is more excellent 50% or more
Select content 65% or more, further preferred content is 75% or more.
Preferably, the molecular weight of the Phellinus fructification cell wall activity Thick many candies is 40k Da-3500k Da, more preferably
Ground is more than 2000k Da.
On the other hand, the present invention also provides a kind of application containing the Phellinus fructification cell wall activity Thick many candies, institutes
Phellinus fructification cell wall activity Thick many candies are stated for improving the immunity of human or animal body.
Compared with prior art, the invention has the following advantages:
Preparation method of the present invention uses the residue after the extraction of Phellinus fructification for raw material, realizes waste circulation
It recycles, is effectively saved preparation cost；Preparation method simple process of the present invention, adds ultramicro grinding step, so that
The even particle size distribution of residue, specific surface area increase, and when extracting step, the contact area of residue and water increases, and effectively mention
The high recovery rate of Thick many candies；Using preparation method of the present invention, it is thin that stable macromolecular Phellinus fructification can be obtained
Cell wall activity Thick many candies, and the content of active Thick many candies is higher, has excellent immunocompetence.
Detailed description of the invention
Fig. 1 is the liquid chromatogram of the Phellinus fructification cell wall activity Thick many candies of embodiment preparation according to the present invention,
The concentration of alcohol that its ethanol precipitation uses is 20%.
Specific embodiment
With reference to the accompanying drawings and examples, further description of the specific embodiments of the present invention.Following embodiment is only
For clearly illustrating technical solution of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment 1
The present embodiment is a kind of preparation method of Phellinus fructification cell wall activity Thick many candies, and its step are as follows:
Step 1: the processing of fructification residue: the residue that fructification water mentions intracellular polyse being crushed with medicinal herb grinder, mistake
20 meshes.Again plus 15 times of water extracts, and 100 DEG C of extraction 3h discard Aqueous extracts.This step 9-10 times repeatedly, to remove water solubility
Intracellular polyse.Phellinus residue drying after water repeatedly is extracted, it is spare.
Step 2: the ultra-fine grinding of Phellinus residue: at -10 DEG C of vibrating grinder that step 1 is residue obtained using pulverizing
15min is crushed, 200 meshes are crossed.The even particle size distribution of residue can be made by crushing material to a few micrometers using this step,
Specific surface area increases, and when extracting step, the contact area of residue and water increases, in favor of the cell wall polysaccharides of macromolecular
Dissolution, effectively increase the recovery rate of Thick many candies.
Step 3: the extraction of cell wall polysaccharides: the water of 15 times of weight being added in the residue after step 2 is pulverized, often
Temperature impregnates 60min, adds distilled water boiling waterbath 3h, and filter residue repeats to extract 2-3 times, combined extract, on a rotary evaporator
Decompression extracting solution is concentrated into material ratio 1:1.2, prepares concentrate；Concentrate is taken, it is insoluble miscellaneous that 10000 × g is centrifuged 30min removal
Matter leaves and takes centrifugate；Three parts of centrifugates are taken to add dehydrated alcohol to alcohol final concentration of 20%, 50%, 70%, quiescent setting respectively
For 24 hours, sediment respectively then is isolated by using 10000 × g centrifugation 20min；Then corresponding alcohol final concentration is used respectively (i.e.
20%, 50%, 70%) ethyl alcohol washs corresponding sediment repeatedly respectively, and corresponding precipitating is finally washed with deionized water respectively
Object carries out the volatilization of ethyl alcohol on water-bath, is dialysed respectively with 8000 molecular weight bag filters (MD34, molecular cut off 8000)
3d removes small-molecule substance, carries out freeze-drying using freeze drier and respectively obtains Phellinus cell wall Thick many candies.
Step 4: the measurement of total sugar content: measuring polyoses content, 20%, 50%, 70% various concentration with sulfuric acid-phynol
Alcohol alcohol precipitation preparation Thick many candies in polyoses content be respectively 86.12%, 53.18%, 57.83%.
Embodiment 2
The present embodiment is the preparation method of another Phellinus fructification cell wall activity Thick many candies, Step 1: step 2
Same as Example 1, step 3 and step 4 are as follows:
Step 3: the extraction of cell wall polysaccharides: the water of 10 times of weight being added in the residue after step 2 is pulverized, often
Temperature impregnates 30min, adds distilled water boiling waterbath 1h, and filter residue repeats to extract 2-3 times, combined extract, on a rotary evaporator
Decompression extracting solution is concentrated into material ratio 1:1.5, prepares concentrate；Concentrate is taken, it is insoluble miscellaneous that 12000 × g is centrifuged 15min removal
Matter leaves and takes centrifugate；Extracting centrifugal liquid adds dehydrated alcohol to alcohol final concentration of 40%, then quiescent setting 30h uses 12000
× g centrifugation 15min isolates sediment；Then with the ethyl alcohol of corresponding alcohol final concentration (i.e. 40%) washing precipitate repeatedly, most
After sediment is washed with deionized water, on water-bath carry out ethyl alcohol volatilization, with 10000 molecular weight bag filters dialyse 4d, removal
Small-molecule substance carries out freeze-drying using freeze drier and respectively obtains Phellinus cell wall Thick many candies.
Step 4: the measurement of total sugar content: measuring polyoses content, the alcohol alcohol precipitation preparation of 40% concentration with sulfuric acid-phynol
Thick many candies in polyoses content be 70.75%.
Embodiment 3
The present embodiment is the preparation method of another Phellinus fructification cell wall activity Thick many candies, Step 1: step 2
Same as Example 1, step 3 and step 4 are as follows:
Step 3: the extraction of cell wall polysaccharides: the water of 12 times of weight being added in the residue after step 2 is pulverized, often
Temperature impregnates 45min, adds distilled water boiling waterbath 2h, and filter residue repeats to extract 2-3 times, combined extract, on a rotary evaporator
Decompression extracting solution is concentrated into material ratio 1:1.3, prepares concentrate；Concentrate is taken, it is insoluble miscellaneous that 8000 × g is centrifuged 30min removal
Matter leaves and takes centrifugate；Extracting centrifugal liquid adds dehydrated alcohol to alcohol final concentration of 60%, quiescent setting 22h, then using 8000 ×
G centrifugation 30min isolates sediment；Then with the ethyl alcohol of corresponding alcohol final concentration (i.e. 60%) washing precipitate repeatedly, finally
Sediment is washed with deionized water, the volatilization of ethyl alcohol is carried out on water-bath, with 9000 molecular weight bag filters dialysis 2d, removes small point
Sub- substance carries out freeze-drying using freeze drier and respectively obtains Phellinus cell wall Thick many candies.
Step 4: the measurement of total sugar content: measuring polyoses content, the alcohol alcohol precipitation preparation of 60% concentration with sulfuric acid-phynol
Thick many candies in polyoses content be 65.53%.
Embodiment 4
The present embodiment is the preparation method of another Phellinus fructification cell wall activity Thick many candies, Step 1: step 2
Same as Example 1, step 3 and step 4 are as follows:
Step 3: the extraction of cell wall polysaccharides: the water of 15 times of weight being added in the residue after step 2 is pulverized, often
Temperature impregnates 60min, adds distilled water boiling waterbath 3h, and filter residue repeats to extract 2-3 times, combined extract, on a rotary evaporator
Decompression extracting solution is concentrated into material ratio 1:1.5, prepares concentrate；Concentrate is taken, it is insoluble miscellaneous that 10000 × g is centrifuged 30min removal
Matter leaves and takes centrifugate；Extracting centrifugal liquid adds dehydrated alcohol to alcohol final concentration of 25%, quiescent setting for 24 hours, then using 8000 ×
G centrifugation 30min isolates sediment；Then with the ethyl alcohol of corresponding alcohol final concentration (i.e. 25%) washing precipitate repeatedly, finally
Sediment is washed with deionized water, the volatilization of ethyl alcohol is carried out on water-bath, with 10000 molecular weight bag filters dialysis 3d, removes small
Molecular substance carries out freeze-drying using freeze drier and respectively obtains Phellinus cell wall Thick many candies.
Step 4: the measurement of total sugar content: measuring polyoses content, the alcohol alcohol precipitation preparation of 25% concentration with sulfuric acid-phynol
Thick many candies in polyoses content be 75.82%.
Embodiment 5
The molecular weight of measurement cell wall polysaccharides is combined using HPSEC-MALLS-RI, the present embodiment uses to be made in embodiment 1
For standby Phellinus cell wall Thick many candies as sample, the concentration of alcohol that ethanol precipitation uses is respectively 20%, 50%, 70%.
Sample pre-treatments: weighing 2mg sample, is dissolved in 1mL mobile phase, is configured to the solution of concentration 2mg/mL.With
Supernatant is taken after 12000 × g centrifugation 10min, HPSEC-MALLS-RI analysis is carried out after 0.25 μm of water phase micro-pore-film filtration.
Analytical column select TSK PWXL6000 and TSK PWXL3000 gel chromatographic columns connect post analysis, mobile phase be containing
The NaH of 0.05mol/L2PO4With the NaNO of 0.15mol/L3Solution (pH=7,0.02% Sodium azide), flow velocity 0.5mL/min,
Chromatographic column temperature column oven is constant at 35 DEG C；Laser detector optical source wavelength selects 623.8nm.The refractive power of polysaccharide in the solution refers to
Number increment (dn/dc) is calculated according to 0.146mL/g.
It is analyzed using Astra (version 6.1.1, Wyatt Technology, Santa Barbara, CA) data soft
Part is acquired and analyzes to light scattering data, calculates molecular weight.20%, the alcohol alcohol precipitation preparation of 50%, 70% various concentration
Obtained Thick many candies middle-molecular-weihydroxyethyl section concentrates on 2500-3500kDa respectively, between 300-600kDa, 40-80kDa.Such as Fig. 1 institute
Show, it is Mw=3159kDa that cell wall Thick many candies main peak molecular weight, which is prepared, in 20% alcohol alcohol precipitation, is ultramicro grinding process
The cell wall polysaccharides of the macromolecular of middle dissolution.
Embodiment 6
To the active research of Phellinus cell wall polysaccharides ion vitro immunization, the present embodiment is thin using the Phellinus prepared in embodiment 1
Cell wall Thick many candies, the concentration of alcohol that ethanol precipitation uses is respectively 20%, 50%, 70%.
The immunocompetence for measuring cell wall Thick many candies is substantially carried out stimulated in vitro macrophage RAW264.7 release NO amount
Test.
NO is easily oxidized in vivo, and forms nitrite, and Griess method measures nitrous acid in culture supernatant
The concentration of salt can reflect macrophage NO burst size indirectly.
Concrete operation step: taking out the cell that ultra low temperature freezer freezes, and 1000rpm is centrifuged 3min, removes frozen stock solution, collects
Cell is evenly distributed in the small culture bottle equipped with DMEM complete medium, is trained under the conditions of 37 DEG C, 5%CO2 saturated humidity
It supports, periodically changes liquid, when cell covers culture bottle bottom of bottle 80~90%, 0.25% trypsin solution heated with 37 DEG C digests, and disappears
Change liquid and be centrifuged 3min in 1000rpm, collect cell, cell is diluted to 5 × 105/mL with colourless 1640 complete medium, counts
Good required hole count is calculated, into 96 porocyte culture plates, 180 μ L cell diluents are added in every hole, 20 μ L samples to be tested are then added,
3 multiple holes are arranged in each sample concentration, and with LPS (10 μ g/mL) for positive control, PBS is negative control, and 96 orifice plates are placed in 37
DEG C, 5%CO248h is cultivated under the conditions of saturated humidity, then draws every 100 μ L of hole supernatant, and 50 μ L Griess reagents are added, put
10min is set, 543nm measures absorbance value.
The result shows that final concentration of 20%, 50%, 70% alcohol precipitation of the alcohol of 100ug/ml obtains Phellinus fructification cell wall
It is respectively 46.05%, 30.16%, 45.16% that Thick many candies stimulating expression of macrophage RAW264.7, which discharges NO amount,.It is final to choose 20%
Alcohol final concentration carry out alcohol precipitation, acquisition Thick many candies activity preferably, close to the burst size 50.14% of positive control LPS, and point
Son amount is big.
Embodiment 7
Analysis to Phellinus cell wall polysaccharides monosaccharide constituent, the present embodiment are thin using the Phellinus prepared in embodiment 1
Cell wall Thick many candies, the concentration of alcohol that ethanol precipitation uses is respectively 20%, 50%, 70%.
Sample pre-treatments: taking Phellinus cell wall Thick many candies sample, accurately weighs 2mg or so with a ten thousandth balance and fills in tool
In test tube, TFA, the 110 DEG C of hydrolysis 4h of 2mol/L are added according to the ratio of (3mL:2mg), it is cooling, it is steamed with revolving instrument low-temperature reduced-pressure
Dry hydrolyzate, is subsequently added into 3mL methanol, evaporated under reduced pressure, and repetitive operation 4-5 times is produced hydrolysis with removing remaining trifluoroacetic acid
Object is cleaned with deionized water is settled to 50mL, and applied sample amount is 20 μ L, with monosaccharide standard Gal, Glu, Ara, Fuc, Rha, Man,
Xyl, Fru, Rib, GluA and GalA are control, with the composition and molar ratio of high-efficiency anion chromatography (HPAEC) measurement monosaccharide.
The result shows that the principal monosaccharides constituent of Phellinus fructification cell wall Thick many candies is glucose, fucose and Portugal
Grape uronic acid, and monosaccharide component molar ratio is 19.70:2.52:1.00, from the above, it can be seen that Phellinus fructification cell wall Thick many candies are main
For macromolecular glucan.
Comparative example 1
Chinese patent CN103319618A discloses a kind of preparation method of active polysaccharide of male agaric mycelium, passes through Phellinus
Then mycelial culture carries out water extraction+ethanol precipitation method and prepares polysaccharide, polysaccharide type include fucose, rhamnose,
Arabinose, galactolipin, glucose, xylose, mannose, the polyoses content finally detected are 16.10%~37.83%.
Comparative example 2
Chinese patent CN105542030A discloses a kind of method that water-soluble beta glucan is extracted from Phellinus fructification,
It prepares Phellinus Thick many candies by lye extraction+ethanol precipitation method, and extracts Phellinus by the method that sour water solution+water redissolves
β water-soluble polysaccharide, the polyoses content finally detected are 50% or more, specially 58%-65%, average molecular weight 2000-
200000 dalton (i.e. 20-200k Da).
Embodiment 1 and comparative example 1, the polyoses content of comparative example 2, average molecular weight and the comparison of polysaccharide type are such as following table institute
Show:
As seen from the above comparison, the Phellinus Thick many candies content prepared by the present invention is apparently higher than comparative example 1, high level
86% is much higher than the 65% of comparative example 2；Phellinus Thick many candies molecular weight prepared by the present invention is also relatively higher than comparative example 1 and comparison
The polysaccharide type of example 2, three's preparation is not also identical.
By above-described embodiment and comparative example it is found that preparation method of the present invention is using residual after the extraction of Phellinus fructification
Slag is raw material, realizes waste cycling and reutilization；Preparation method of the present invention exists to sub- solid-cell wall middle-molecular-weihydroxyethyl
2000000 or more macromolecular polysaccharide is excavated, and the further development and utilization of rare Resources of Medicinal Fungi are facilitated, gained
Polyoses content 50% or more and up to 75% or more, the polysaccharide molecular weight of preparation is between 40-3500k Da, mainly
For stable macromolecular glucan.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it any equivalent modifications to the practical progress and replaces
In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and repair
Change, all should be contained within the scope of the invention.
Claims (5)
1. a kind of preparation method of Phellinus fructification cell wall activity Thick many candies, which comprises the steps of:
Step 1: the processing of fructification residue: the residue that fructification water mentions intracellular polyse being crushed with medicinal herb grinder, is sieved；
Add boiling water after water soak at room temperature to extract repeatedly, discards Aqueous extracts, the mulberry to remove water-soluble intracellular polyse, after water repeatedly is extracted
Yellow residue drying, it is spare；
Step 2: the ultra-fine grinding of Phellinus residue: the resulting Phellinus residue of step 1 is crushed using vibrating grinder is pulverized,
Sieving；
Step 3: the extraction of Phellinus fructification cell wall activity Thick many candies: adding in the Phellinus residue after step 2 is pulverized
Entering the water of 10-15 times of weight, soak at room temperature 30-60min adds distilled water boiling waterbath 1-3h, and filter residue repeats to extract 2-3 times,
Combined extract；It is 1:1.2-1.5 that the extracting solution to material ratio is concentrated under reduced pressure on a rotary evaporator, prepares concentrate；It takes
Concentrate, 8000-12000 × g centrifugation removal 15-30min insoluble impurities, leaves and takes centrifugate；In centrifugate plus ethyl alcohol is heavy
Shallow lake 20h or more, sediment separate out wash sediment repeatedly using alcohol identical with ethanol precipitation concentration, are then made
Sediment is dissolved with water, with 8000-10000 molecular weight bag filter dialysis 2-4d, removes the small-molecule substance in sediment, finally
The sediment of removal small-molecule substance is freeze-dried, to obtain Phellinus fructification cell wall activity Thick many candies；
Wherein, for the alcohol concentration that the ethanol precipitation uses for 20%, the molecular weight of activity Thick many candies obtained is 2500-
3500kDa；The Phellinus fructification cell wall activity Thick many candies are mainly macromolecular glucan；
Wherein, the operating time that vibrating grinder is pulverized in the step 2 is 10~20min, and operation temperature is -10 DEG C.
2. preparation method according to claim 1, which is characterized in that further include thick more to Phellinus fructification cell wall activity
The step of sugar is further purified.
3. preparation method according to claim 1, which is characterized in that the mesh number being sieved in the step 1 is 20 mesh, institute
Stating the mesh number being sieved in step 2 is 200 mesh.
4. preparation method according to claim 1, which is characterized in that the Phellinus fructification cell wall activity Thick many candies
Content is 50% or more.
5. a kind of application of the Phellinus fructification cell wall activity Thick many candies of preparation method preparation according to claim 1,
It is characterized in that, the application is that Phellinus fructification cell wall activity Thick many candies are being prepared for improving the immune of human or animal body
Application in the preparation of power.
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| CN111019008B (en) * | 2019-12-13 | 2022-04-05 | 浙江省农业科学院 | Anti-inflammatory activity phellinus igniarius polysaccharide SHP and preparation method thereof |
| CN114044832B (en) * | 2021-12-15 | 2023-06-23 | 中华全国供销合作总社济南果品研究所 | Method for extracting Phellinus linteus fruiting body polysaccharide |
| CN116217748B (en) * | 2023-03-01 | 2024-05-14 | 宁波御菌生物技术有限公司 | Method for extracting polysaccharide from Phellinus linteus fruiting body |
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